Dr Aleksandar Ivetic BSc (Hons) ARCS PhD
King's College London
James Black Centre
125 Coldharbour Lane
London SE5 9NU
Dr Ivetic was appointed Senior Lecturer in Cardiovascular Biology in April 2009 and is Principal Investigator of the Membrane/Cytoskeleton Signalling Group. Dr Ivetic graduated from Imperial College London with a BSc (Hons) in 1993 in Biochemistry and additionally became an Associate of the Royal College of Science (ARCS). He then moved to the Marie Curie Research Institute (Oxted, Surrey), where, in association with the Institute of Cancer Research (University of London), he embarked on a PhD in Biochemistry to understand how DNA replication was controlled by the Cell Cycle.
His first post-doctoral position was held at the National Institute for Medical Research (NIMR, Mill Hill, London) where he applied his expertise in protein biochemistry to the field of leukocyte trafficking. He then moved to the Ludwig Institute for Cancer Research (UCL branch, London) for his second postdoctoral position, where he gained extensive knowledge in cell biology, with a main focus on leukocytes, fibroblasts and endothelial cells. He subsequently moved to Imperial College London (National Heart and Lung Institute) in July 2005 as part of a Wellcome Trust Research Career Development Award, which enabled him to continue his interests in leukocyte trafficking, and to develop skills to analyse leukocyte adhesion under flow conditions.
Leukocyte trafficking in cardiovascular disease – the big goal
Inflammatory cells (such as neutrophils, monocytes and lymphocytes) are known to drive the progression of many chronic and acute cardiovascular diseases such as atherosclerosis, myocardial injury induced by ischemia and cardiac allograft rejection. Therefore, understanding the detailed molecular mechanisms by which leukocytes are recruited to sites of tissue injury within the cardiovascular system will provide the potential to develop therapeutic strategies to curb acute and chronic inflammatory disorders that plague tissues and organs during disease progression.
Below in Movie 1, neutrophils are seen to “swarm” toward a site of non-infectious “sterile injury”. We have used a high-intensity laser beam directed at a piece of tissue, so that we can track the fate of neutrophils migrating from blood vessels towards the site of injury. Sterile injury underpins acute and chronic inflammatory states within the cardiovascular system, and currently there is little known about the molecular differences between infectious and non-infectious injury. Neutrophils are excellent at killing infectious agents, because they can track down the source of infection very rapidly and deploy numerous toxic substances to ensure the infection doesn’t spread to the rest of the body. Unfortunately neutrophils use a similar strategy during sterile injury, which adds more harm than good - for example in the context of myocardial infarction. We want to know what the molecular events are that lead to unwanted aggressive neutrophil-led damage during sterile injury.
Movie 1 In collaboration with Dr. Paul Kubes (University of Calgary, Canada) we are using intravital microscopy, to identify molecular changes occurring in neutrophils as they swarm to a site of sterile (laser) injury. Note in the movie: neutrophils close to blood vessels carry both red and blue signals at the start of the movie. As the movie progresses, the red signal is lost from neutrophils as they arrive to the site of sterile injury. We are interested in defining the molecular mechanism that mediates such molecular changes in order to better understand sterile inflammation.
The Membrane/Cytoskeleton Signalling Group
Our primary research aim is to understand the molecular mechanisms that facilitate leukocyte adhesion to the luminal walls of blood vessels. Binding of leukocytes to the vessel wall is an absolute requirement for successful passage out of the vasculature and in to the surrounding tissue. This process is embodied by the “leukocyte multi-step adhesion cascade” (Figures 1 & 2), which is crudely broken down in to:
- Initial capture (or tethering)
- Firm adhesion
A number of different cell adhesion molecules on the surface of leukocytes are responsible for mediating tethering and rolling. One such molecule is called L-selectin, which is presented on the tips of finger-like projections called microvilli. These microvilli provide L-selectin with a biological advantage to recognise its ligand over other cell adhesion molecules present on the plasma membrane. The cortical actin-based cytoskeleton is an essential structural scaffold that is required for the formation of microvilli, ensuring the correct anchoring of L-selectin to microvilli.
Figure 1 - Simplified overview of the multi-step leukocyte adhesion cascade (Courtesy of Marouan Zarrouk and Rufus Ho). A local inflammatory insult leads to signalling events that stimulate endothelial cells within the local vicinity to up-regulate cell adhesion molecules that promote leukocyte recruitment. The L-selectin on bystander leukocytes comes in contact with glycans presented by underlying endothelial cells in the form of sialyl Lewis x (sLex). Low affinity bond formation between L-selectin and sLex promotes tethering. Cell tethering is only translated into rolling when the underlying ligand is in sufficient abundance to mediate the continuity of bond formation at the front of the cell and bond breakage at the back of the cell. Cell rolling is critical for endothelial cells to communicate an “arrest” signal to the leukocytes, which comes in the form of chemokines. Leukocytes finally exit the vasculature by transmigrating either through or between endothelial cells, where they begin to follow gratients of other chemoattractants deep in the tissue.
Figure 2 - Neutrophil transmigration through an endothelial cell monolayer Neutrophils are perfused over TNF-activated human umbilical vein endothelial cells (HUVEC) and tracked over time by timelapse brightfield microscopy. Note that adherent neutrophils on top of HUVECs are “phase bright” and transmigrated neutrophils appear “phase dark”. Note also that dramatic changes in cell shape also occur during the transmigration phase.
We discovered the ezrin-radixin-moesin (ERM) family of proteins as binding partners of the short, 17 amino acid, cytoplasmic tail of L-selectin. ERMs essentially link the cortical actin cytoskeleton with the plasma membrane by generating direct interactions via their C- and N-termini (Figure 3). Overexpression of cell adhesion molecules that bind ERM (such as L-selectin) can promote the formation of microvilli, and knocking down expression of ERMs can lead to the loss of microvilli. We have shown that abrogating L-selectin/ERM interaction reduces microvillar positioning of L-selectin, which in turn affects leukocyte tethering to immobilised ligand under conditions of flow (using in vitro flow chamber assays). Collectively, these observations suggest an inter-dependent relationship between L-selectin, microvilli and ERMs, the mechanism of which is poorly understood.
Figure 3 - Schematic model of how ERM proteins are regulated
In their inactive states, the N- (red clover-leaf shape) and C- (green oval) termini interact with one another to form either an auto-inhibited “closed” conformation. Others have reported that ERMs can bind in an anti-parallel fashion. In either case, it is the interaction between N- and C-termini that masks binding sites for interaction with other proteins. Phosphorylation of a conserved threonine residue results in the opening of the closed conformation. This enables the N- and C-termini to bind to the tails of cell adhesion molecules (such as L-selectin) and the cortical actin cytoskeleton, respectively. ERMs can also flip open by binding to PIP2, a phospholipid found in the inner leaflet of the plasma membrane. This mode of ERM activation appears to be more prevelant in leukocytes, with C-terminal phosphorylation acting as a stabilising effect on the open molecule.
We have taken further steps to understand how such a short cytoplasmic tail can accommodate the binding of more than one partner and how this might be involved in mediating signal transduction during adhesion (Figure 4). Indeed, others have shown that clustering of L-selectin (which is thought to occur during L-selectin-dependent tethering and rolling) can promote mobilisation of the chemokine receptor, CXCR4, to the plasma membrane and (β1 and β2) integrin activation. Both of these events are necessary for progression through the adhesion cascade, and signalling through L-selectin is therefore though to be involved in mediating the transition from rolling to arrest – either independently of, or in concert with, chemokines.
Figure 4 - Possible signalling mechanisms during cell rolling
(A) Molecular modelling reveals that the short cytoplasmic tail of L-selectin can accommodate the binding of both calmodulin and ERM (e.g. N-terminal moesin, or FERM). We have recently shown, using fluorescence lifetime imaging microscopy (FLIM) to monitor fluorescence resonance energy transfer (FRET), that calmodulin and ERM interact with one another in intact cells (B), which supports our previous in vitro and in silico findings (see reference 1 for more information). This implies that remodelling of the extracellular domains of L-selectin (through ligand binding) leads to changes in how L-selectin complexes with its cytosolic binding partners (C). Such remodelling events could be involved in signalling during cell tethering and rolling and therefore facilitate the transition from rolling to arrest.
A new role for L-selectin in regulating transendothelial migration
Unlike its family members, E- and P-selectin, L-selectin contains a unique membrane-proximal extracellular cleavage site. L-selectin is expressed on most circulating leukocytes, but is rapidly “shed” in response to cell-activating stimuli. Ectodomain shedding regulates the surface levels of L-selectin, which is one way in which intracellular signals can be rapidly shut down. Very recently we have shown that ectodomain shedding of L-selectin is triggered in CD14-positive monocytes specifically during transendothelial migration – and not before (see movie 2). We realised the biological importance of L-selectin (in its non-cleaved form) during transmigration was to potentiate “invasion” across activated endothelial monolayers. The timing of ectodomain shedding of L-selectin was essential to establish front-back polarity in the fully transmigrated cells. We wish to explore if this phenomenon can be exploited in the setting of sterile inflammation. Currently, we are building bespoke flow chambers to address the impact of blocking L-selectin in regulating leukocyte invasion and chemotaxis through 3D collagen scaffolds (see Figure 5).
CD14-positive monocytes are perfused over TNF-activated HUVEC and allowed to undergo the full gamut of the multi-step adhesion cascade. In the right panel, monocytes have been labelled with a fluorescently tagged anti-L-selectin-specific antibody (LAM1-14). Note that this signal is lost specifically when monocytes undergo transmigration.
Figure 5 - By growing endothelial cell monolayers on top of a 100 micron-thick bed of type I collagen, we are able to perfuse leukocytes over activated endothelial cells and allow them to transmigrate towards a chemoattractant source that is fed from microfluidic channels positioned directly beneath the collagen bed. The lower image is a still of the movie showing the fate of leukocytes in 2 phases: the first is perfusion over activated endothelial cells and the second is their chemotaxis towards the chemoattractant source. This work is performed in collaboration with Dr. Guillaume Charras (UCL).
- Molecular Biology
- Protein Biochemistry
- Cell Biology
- In vitro flow assays (fluorescence timelapse video microscopy)
- Intravital microscopy
- Mr Ross King
- Ms Abigail Newe
- Ms Karolina Rzeniewicz
- Ms Hannah Tomlins
From left to right: Ms Abigail Newe, Dr Angela Rey Gallardo, Ms Karolina Rzeniewicz, Ms Hannah Tomlins
- Rzeniewicz K, Newe A, Rey Gallardo A, Davies J, Holt MR, Patel A, Charras GT, Stramer B, Molenaar C, Tedder TF, Parsons M, Ivetic A. L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro. Proc Natl Acad Sci U S A. 2015 Mar 24;112(12):E1461-70.
- Murdoch CE, Chaubey S, Zeng L, Yu B, Ivetic A, Walker SJ, Vanhoutte D, Heymans S, Grieve DJ, Cave AC, Brewer AC, Zhang M, Shah AM. Endothelial NADPH oxidase-2 promotes interstitial cardiac fibrosis and diastolic dysfunction through proinflammatory effects and endothelial-mesenchymal transition. J Am Coll Cardiol. 2014 Jun 24;63(24):2734-41.
- Murugaesu N, Iravani M, van Weverwijk A, Ivetic A, Johnson DA, Antonopoulos A, Fearns A, Jamal- Hanjani M, Sims D, Fenwick K, Mitsopoulos C, Gao Q, Orr N, Zvelebil M, Haslam SM, Dell A, Yarwood H, Lord CJ, Ashworth A, Isacke CM. An in vivo functional screen identifies ST6GalNAc2 sialyltransferase as a breast cancer metastasis suppressor. Cancer Discov. 2014 Mar;4(3):304-17.
- Patel AS, Smith A, Nucera S, Biziato D, Saha P, Attia RQ, Humphries J, Mattock K, Grover SP, Lyons OT, Guidotti LG, Siow R, Ivetic A, Egginton S, Waltham M, Naldini L, De Palma M, Modarai B. TIE2-expressing monocytes/macrophages regulate revascularization of the ischemic limb. EMBO Mol Med. 2013 Jun;5(6):858-69.
- Ivetic A. Signals regulating L-selectin-dependent leucocyte adhesion and transmigration. Int J Biochem Cell Biol. 2013 Mar;45(3):550-5.
- Burns SO, Killock DJ, Moulding DA, Metelo J, Nunes J, Taylor RR, Forge A, Thrasher AJ, Ivetic A. A congenital activating mutant of WASp causes altered plasma membrane topography and adhesion under flow in lymphocytes. Blood. 2010 Jul 1;115(26):5355-65.
- Killock DJ, Ivetic A. The cytoplasmic domains of TNFalpha-converting enzyme (TACE/ADAM17) and L-selectin are regulated differently by p38 MAPK and PKC to promote ectodomain shedding. Biochem J. 2010 May 13;428(2):293-304.
- Killock DJ, Parsons M, Zarrouk M, Ameer-Beg SM, Ridley AJ, Haskard DO, Zvelebil M, Ivetic A. In Vitro and in Vivo Characterization of Molecular Interactions between Calmodulin, Ezrin/Radixin/Moesin, and L-selectin. J Biol Chem. 2009 Mar 27;284(13):8833-45.
- Erwig LP, McPhilips KA, Wynes MW, Ivetic A, Ridley AJ, Henson PM. Differential regulation of phagosome maturation in macrophages and dendritic cells mediated by Rho GTPases and ezrin-radixin-moesin (ERM) proteins. Proc Natl Acad Sci U S A. 2006 Aug 22;103(34):12825-30.
- Ivetic A*, Ridley AJ. The telling tail of L-selectin. Biochem Soc Trans. 2004 Dec;32(Pt 6):1118-21.
- Ivetic A*, Florey O, Deka J, Haskard DO, Ager A, Ridley AJ. Mutagenesis of the ezrin-radixin-moesin binding domain of L-selectin tail affects shedding, microvillar positioning, and leukocyte tethering. J Biol Chem. 2004 Aug 6;279(32):33263-72.
- Ivetic A, Ridley AJ. Ezrin/radixin/moesin proteins and Rho GTPase signalling in leucocytes. Immunology. 2004 Jun;112(2):165-76.
- Ivetic A*, Deka J, Ridley A, Ager A. The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin. J Biol Chem. 2002 Jan 18;277(3):2321-9.
* = corresponding author